Development of the first potent and specific inhibitors of endocannabinoid biosynthesis

Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1- selective-diacylglycerol lipases a and ß (DAGLa and ß) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLa to screen new synthetic substances as DAGLa inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLa and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2- arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat®) (IC50~60 nM), the most potent inhibitors of DAGLa were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50=500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50=160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat Nacylphosphatidyl- ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitrophenyl oleylphosphonate] inhibited both DAGLa and MAGL with similar potencies (IC50=0.8-0.1 and 3.7-3.2 µM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.